Pre- and post-harvest inhibition of remobilisation of storage compounds

ABSTRACT

This invention describes a method to prevent sprouting in vegetatively propagated plants such as potato, strawberry, banana and bulbous plants such as onion and bulbous flowers, by transforming a plant or a plant from one of its parental lines with a gene coding for trehalose phosphate synthase. Restoration of sprouting is also provided for.

FIELD OF THE INVENTION

[0001] This application is concerned with the pre- and postharvestinhibition of remobilisation of storage compounds. Especially, theapplication describes the prevention of sprouting, especially invegetatively propagated plants by transforming them with recombinant DNAand a method to restore sprouting in these lines.

BACKGROUND ART

[0002] In traditional breeding as well as in agricultural geneticengineering the major goal is to obtain crops with a high yield, whichgenerally means that the goal has been to increase storage of the plantin the organs of the plant that are used for storage, such as the tubersin potato, the taproot in sugarbeet, and the leaves in leafy crops suchas lettuce. However, other processes in plants, such as flowering and orsprouting, often give a yield penalty.

[0003] Sprouting normally can be inhibited by cold storage at very lowtemperatures (slightly above freezing). Cold storage is not onlyexpensive, but also inflicts deleterious effects upon storage organs,which render them unsuitable for further processing or result in yieldlosses of commercial products as starch For example when potato tubersare subjected to cold temperatures, they convert starch to reducingsugars, a phenomenon known as ‘cold sweetening’. The development ofreducing sugars is very undesirable because during baking and fryinge.g. the Maillard reaction occurs that results in undesired browning.

[0004] To prevent cold sweetening potatoes can be stored at highertemperatures, but this results in undesired sprouting. Amongst others,chlorpropham (CIPC) is used by the industry to control tuber sprouting.Although CIPC has been used effectively, it still is considered as anundesirable chemical treatment. All around the world, there is anincreasing emphasis on replacing chemical control agents with biologicalcontrol mechanisms that are safe and more environmentally acceptable.

[0005] When considering a genetic approach to inhibit sprouting, it mustalso be considered that for the development of see-potatoes sprouting isa desired property, and that thus a mechanism should be at hand whichenables seed-potato production but which prevents sprouting in potatoescultured for consumption or further processing.

SUMMARY OF THE INVENTION

[0006] This invention comprises a method to inhibit pre- and postharvestremobilisation of storage compounds. More specifically, the inventioncomprises a method to prevent sprouting of a plant part by transformingthe plant or its ancestor with a recombinant DNA capable of expressionof a protein, characterized in that the protein is trehalose phosphatesynthase (TPS). More specifically the recombinant DNA comprising thegene coding for TPS is of bacterial, fungal, animal, plant or humanorigin, preferably derived from Escherichia coli.

[0007] In another embodiment the invention comprises a method to inducesprouting in a plant by providing said plant with recombinant DNA codingfor TPS flanked by target sites of a site-specific recombinase andremoving the recombinant DNA coding for TPS by providing said planteither through transformation with a gene coding for the correspondingrecombinase or through crossing with a plant capable of expressing saidrecombinase.

[0008] Still another embodiment of the invention comprises a method toinduce sprouting in a plant by providing a plant with recombinant DNAcoding for TPS and subsequently or simultaneously transforming it with arecombinant DNA which comprises a gene coding for a molecule that canneutralize the effect of TPS under control of an inducible promoter andforcing expression of the neutralizing molecule by induction of theinducible promoter. An example of such a neutralizing molecule istrehalose phosphate phosphatase (TPP) or the product of the antisenseTPS gene.

[0009] Another embodiment of the invention is formed by removing theinhibition of pre- and post-harvest mobilisation of storage compounds byexternal treatment with compounds that neutralize the inhibitory effectof the expression of the TPS gene. Preferably this is accomplished byapplying gibberellic acid. Still another embodiment of the invention isto restore sprouting by wounding.

[0010] A further object of the invention is a method to induce sproutingin a plant by providing a plant with recombinant DNA coding for TPS andsubsequently or simultaneously transforming it with a recombinant DNAwhich comprises a gene coding a suppressor under control of an induciblepromoter, said suppressor capable of suppressing expression of the TPSand forcing expression of the suppress or by induction of the induciblepromoter.

[0011] Also the invention provides for plants made by any of the abovementioned methods, specifically vegetatively propagated plants and morespecifically potato and onion.

[0012] Further the gene coding for TPS can be placed under control of aspecific promoter, such as the patatin promoter, which specificallygives expression in the tuber of the potato plant.

[0013] Another embodiment of the invention is the inhibition of thecatabolism of inulin in chicory, the inhibition of sucrose catabolism insugarbeet and the inhibition of starch degradation in potato.

DESCRIPTION OF THE FIGURES

[0014]FIG. 1. Sprouting behaviour of patatin-TPS tubers with or withouttreatment with gibberellic acid (GA) after 14 days (FIG. 5A) and after25 days (FIG. 5B).

DETAILED DESCRIPTION OF THE INVENTION

[0015] For definition purposes only the general term of a transformedplant is a plant totality or a plant grouping. This term is meant tocover a broad spectrum of plants and is not confined to one specificvariety.

[0016] The invention is concerned with a met hod for the pre- and/orpostharvest inhibition of remobilisation of storage compounds. Theremobilisation of storage compounds is the process that plants undertaketo utilise the compounds that have been stored, generally in specialisedstorage organs. A typical example of such a mobilisation is the processof sprouting from storage organs such as tubers, bulbs or seeds.

[0017] Specifically, provided are methods for the inhibition ofsprouting, preferably in vegetatively propagated plants and methods torestore sprouting capabilities again in plants that have been inhibited.Sprouting in this sense is defined as the formation of shoots, runners,stolons or suckers, especially from storage tissue

[0018] The basis of this invention is found in the fact that it has beensurprisingly found that expression of TPS inhibits sprouting. TPS is anenzyme which is active in the trehalose synthesis pathway, which is notpresently known to play a role in sprouting tissue. However, it has beenrecently found (WO 97/42326) that the enzymes TPS and TPP are able tochange dramatically the carbohydrate metabolic and photosyntheticcapacity of tissues in which they are expressed. It has furthermore beenfound that the effects of TPP and TPS are opposite, i.e. by simultaneousexpression no major effects on the plant physiology and phenotype can beobserved. In said application it has additionally been found that byexpressing TPS in the tuber also the effects of the ‘cold sweetening’process can be diminished, because the proportion of reducing sugars isdecreased at harvesting and after storage. Thus, taking also into regardthe present invention, expression of TPS may improve the storage ofpotatoes in two ways: for cold storage the effect of diminishing thecold sweetening process is important, while for storage under moremoderate temperature the prevention of sprouting prevails.

[0019] Thus, TPS is capable to prevent remobilisation of storagecompounds. This is also applicable in other crops, such as chicory,which is subject to degradation of the inulin into other carbohydrates.Expression of TPS in the storage organs of chicory prevents catabolicdegradation of the inulin. Similarly, sucrose breakdown in sugarbeet canbe prevented. Thus, expression of TPS in the taproots of sugarbeetprevents the loss of sucrose during storage of the sugarbeets.

[0020] Generally, the anti-sprouting effect is obtained by theexpression of the TPS gene preferably in the tissues which are prone tosprouting, such as the potato tuber. For specific expression in thepotato tuber the patatin promoter or any other tuber-specific promotermay be used to drive the expression of the TPS gene. We have, however,noted that it is most important that the promoter is active at the endof the filling phase of the tuber and during storage of the tuber. Ifthe tuber-specific promoter is not very active anymore at that point,the inhibitory effects of the expression of TPS will wane off, and adelay in sprouting in stead of a complete inhibition of sprouting willbe the result.

[0021] The TPS gene is encoding a trehalose phosphate synthase. Severalgenes coding for this enzyme are known and can be found in all kind oforganisms (WO 97/42326). In the experiments sustaining the invention thegene derived from Escherichia coli is used, but also other genes codingfor TPS, e.g. derived from yeast or plants, are equally useful. In otherembodiments of the invention compounds neutralizing the effect of TPSsuch as trehalose phosphate phosphatase (TPP) are used. Also the genecoding for TPP is derived from E. coli, but it can equally well bederived from other organisms such as yeast, plants or even humans (WO97/42326). Not only the TPP is useful to restore the effects of TPS butany enzyme capable of degrading trehalose-6-phosphate can be used. Afurther example of such an enzyme is trehalose-6-phosphate hydrolase(TreC). A gene coding for this enzyme can be drived from E. coli(Rimmele, M., and Boos, W., Trehalose-6-phosphate hydrolase ofEscherichia coli. J. Bacteriol. 176, 5654-5664, 1994).

[0022] In its simplest form the invention is directed to inhibit pre-and postharvest remobilisation of storage compounds in a transgenicplant by transforming plant with a recombinant DNA cassette whichcomprises the gene coding TPS and optionally a selectable marker gene.More specifically such a method prevents sprouting. Restoration ofsprouting can be obtained by neutralizing the effect of TPS. This can beachieved in a number of ways. The following are given by example butmethods to inhibit the effect of TPS are not limited to these examples.

[0023] A first system of restoration of sprouting is to introduce nextto the TPS gene a gene coding for TPP, which is able to overcome theanti-sprouting effects caused by the TPS. To prevent the constitutiveexpression of TPP it is envisaged to bring expression of TPP undercontrol of an inducible promoter. Inducible promoters include anypromoter capable of increasing the amount of gene product produced by agiven gene, in response to exposure to an inducer. In the absence of aninducer the DNA sequence will not be transcribed. Typically, the factorthat binds specifically to an inducible promoter to activatetranscription is present in an inactive form which is then directly orindirectly converted to the active form by the inducer. The inducer maybe a chemical agent such as protein, metabolite (sugar, alcohol, etc.),a growth regulator, herbicide, or a phenolic compound or a physiologicalstress imposed directly by heat, salt, wounding, toxic elements etc., orindirectly through the action of a pathogen or disease agent such as avirus. A plant cell containing an inducible promoter may be exposed toan inducer by externally applying the inducer to the cell such as byspraying, watering, heating, or similar methods. Inducible promoters areknown to those familiar with the art and several exist that couldconceivably be used to drive expression of the TPP gene. Induciblepromoters suitable for use in accordance with the present inventioninclude, but are not limited to, the heat shock promoter, the mammaliansteroid receptor system and any chemically inducible promoter. Examplesof inducible promoters include the inducible 70 kD heat shock promoterof Drosophila melanogaster (Freeling, M. et al., Ann. Rev. Genet. 19,297-323) and the alcohol dehydrogenase promoter which is induced byethanol (Nagao, R. T. et al., in: Miflin, B. J. (ed.) Oxford Surveys ofPlant Molecular and Cell Biology, Vol. 3., pp. 384-438, Oxford Univ.Press, 1986). A promoter that is inducible by a simple chemical isparticularly useful. Examples for the last category are the promotersdescribed in WO 90/08826, WO 93/21334, WO 93/031294 and WO 96/37609.

[0024] Thus, the anti-sprouting effect can be restored by treatment withthe inducer, and these restored sprouting lines can be used to propagatethe seeding material, such as seed-potatoes. Without the presence of theinducer, sprouting of the offspring is still inhibited by the expressionof TPS. This thus also functions as a way to produce germplasmprotection.

[0025] A further method to restore the original sprouting phenotypeagain is to provide the plant with a recombinant DNA cassette whichcomprises next to the TPS gene an antisense TPS gene, said antisensegene being under control of an inducible promoter As with theabove-mentioned example on the induction of TPP also the antisense TPSis capable of negating the effect of the (sense) TPS expression becauseby annealing with the TPS mRNA it prevents successful translation of theTPS and thus inhibits the anti-sprouting effect.

[0026] A third system of restoration of the original sprouting phenotypeis by introducing the DNA coding for a suppressor protein, saidsuppressor capable of suppressing the expression of TPS, while theexpression of the suppressor is under control of and inducible promoter.Such a suppression can for instance be accomplished by use of thetet-repressor system, where a specific binding site, which can berecognized by the repressor, is introduced near the RNA-polymerasebinding site of the gene which expression needs to be suppressed. If thetet-repressor is available then this repressor will bind to the specificsequence and thus, by steric hindrance, prevents the RNA-polymerase toinitiate transcription. The gene coding for the tet-repressor can beadjacent the gene which expression should be controlled, but this is notnecessary.

[0027] When the gene for the repressor is put under control of aninducible promoter the expression of the suppressor-molecule and thusthe suppression of the TPS gene can be induced by applying an externalinducer. Then, the TPS effect will not be established and normalsprouting will be the result.

[0028] A further system to restore the normal phenotype is to providethe gene coding for TPS or the expression cassette comprising said geneflanked by two site-specific recombination sites, which can berecognized by the corresponding recombinase.

[0029] A number of different site-specific recombinase systems can beutilized in accordance the present invention, including but not limitedto the Cre/lox system of bacteriophage P1, the FLP/FRT system of yeast,the Gin recombinase of phage Mu, the Pin recombinase of E. coli, and theR/RS system of the pSR1 plasmid. The two most used site-specificrecombinase systems are the bacteriophage P1 cre/lox and the yeastF(P/FRT systems. In these systems a recombinase (Cre or FLP) interactsspecifically with its respective site-specific recombination sequence(lox or FRT, respectively) to invert or excise the interveningsequences. The site-specific recombination sequence for each of thosetwo systems is relatively short (34 bp for lox and 34-47 bp for FRT).Use of such a site-specific recombinase in plants is for instancedescribed in U.S. Pat. No. 5,527,695. The DNA to be excised can beflanked by direct repeats of the site-specific recombination site, andsubsequent introduction of the recombinase activity excises the DNA (andthus restores the original phenotype). The FLP/FRT recombinase systemhas been demostrated to function efficiently in plant cells. Althoughthe site-specific recombination sequences must be linked to the ends ofthe DNA sequence to be excised for inverted, the gene encoding thesite-specific recombinase may be located elsewhere and thus can beseparately introduced into the plant cells through standardtransformation procedure, or through cross-pollination with a plant thatalready is capable of expressing the recombinase gene.

[0030] However, upon this last method of restoration the TPS gene islost from he transgenic plants.

[0031] Other ways to remove the inhibitory effects of the expression ofthe TPS gene on the remobilisation of storage compounds are externaltreatments of the storage organs with compounds that are capable ofneutralizing the effects of the expression of the TPS gene.Surprisingly, we have found that treatment with gibberellic acid (GA)was able to induce sprouting in potato tubers contain the TPS gene. Thiswas accomplished by incubation of whole tubers or cut pieces in asolution of commercially available GA. It is, however, envisaged thatthe method of treatment can be varied and that for instance spraying oftubers with a GA solution would yield comparable results. Depending onthe way of application the concentration of GA in the solution should bein the range of 0.1 to 10,000 ppm. It is further believed that theeffect of GA is a neutralization of the effects of expression of the TPSgene. Therefor, it is envisaged that also in other examples ofinhibition of remobilisation of storage compounds, treatment with GAwill be able to restore the inhibitory effects of the expression TPS.

[0032] Also surprisingly, we have found that wounding of potato tubers(through cutting off pieces containing at least one active meristem)alone was sufficient to induce sprouting of those pieces.

[0033] The recombinant DNA constructs of the present invention can beconstructed using recombinant DNA technology known to those skilled inthe art. The recombinant gene constructs can be inserted into vectors,which can be commercially available, specifically suited fortransformation to plants and to express the gene product in thetransformed cells. Transformed cells (those containing the recombinantDNA inserted into the host cell's DNA) are selected from untransformedcells through the use of a selectable marker included as part of theintroduced recombinant DNA. Selectable markers include genes thatprovide antibiotic or herbicide resistance. Those cells containing therecombinant DNA are capable of surviving in the presence of antibioticor herbicide concentrations that kill untransformed cells. Examples ofselectable marker genes include the bar gene which provides resistanceto the herbicide Basta, the nptII gene which confers kanamycinresistance, the hpt gene which confers hygromycin resistance and the cahgene which gives resistance to cyanamid. An entire plant can begenerated from a single transformed plant cell through cell culturingtechniques known to those skilled in the art.

[0034] With regard to the applicability of the invention in differentplant species, it has to be mentioned that one particular embodiment ofthe invention is merely illustrated with transgenic potato plants as anexample, the actual applicability being in fact not limited to thisplant species. Any plant species can be provided with a recombinant DNAsequence according to the invention, but preferably plant species whichare normally vegetatively propagated are especially useful.

[0035] Although some of the embodiments of the invention may not bepracticable at present, e.g. because some plant species are as yetrecalcitrant to genetic transformation, the practicing of the inventionin such plant species is merely a matter of time and not a matter ofprinciple, because the amenability to genetic transformation as such isof no relevance to the underlying embodiment of the invention.

[0036] Transformation of plant species is now routine for an impressivenumber of plant speicies, including both the Dicotyledoneae as well asthe Monocotyledoneae. In principle any transformation method may be usedto introduce recombinant DNA according to the invention into a suitableancestor cell, as long as the cells are capable of being regeneratedinto whole plants. Methods may suitably be selected from thecalcium/polyethylene glycol method for protoplasts (Krens, F. A. et al.,1982, Nature 296, 72-74; Negrutiu I. et al, June 1987, Plant Mol. Biol.8, 363-373), electroporation of protoplasta (Shillito R. D. et al., 1985Biol/Technol. 3, 1099-1102), microinjection into plant material(Crossway A. et al., 1986, Mol. Gen. Genet. 202, 1719-185), (DNA orRNA-coated particle bombardment of various plant material (Klein T. M.et al., 1987, Nature 327, 70), infection with (non-integrative) virusesand the like. A preferred method according to the invention comprisesAgrobacterium-mediated DNA transfer. Especially preferred is the use ofthe so-called binary vector technology as disclosed in EP A 120 516 andU.S. Pat. No. 4,940,838). Tomato transformation can be preferably doneessentially as described by Van Roekel et al. (Van Roekel, J. S. C.,Damm, B., Melchers, L. S., Hoekema, A. (1993). Factors influencingtransformation frequency of tomato (Lycopexsicon esculentum). Plant CellReports, 12, 644-647). Potato transformation can be preferably doneessentially as described by Hoekema et al. (Hoekema, A., Huisman, M. J.,Molendijk, L., van den Elzen, P. J. M., and Cornelissen, B. J. C.(1989). The genetic engineering of two commercial potato cultivars forresistance to potato virus X. Bio/Technology 7, 273-278). Generally,after transformation plant cells or cell groupings are selected for thepresence of one or more markers which are encoded by plant expressiblegenes co-transferred with the nucleic acid sequence encoding the proteinaccording to the invention, whereafter the transformed material isregenerated into a whole plant.

[0037] Although considered somewhat more recalcitrant towards genetictransformation, monocotyledonous plants are amenable to transformationand fertile transgenic plants can be regenerated from transformed cellsor embryos, or other plant material. Presently, preferred methods fortransformation of monocots are microprojectile bombardment of embryos,explants or suspension cells, and direct DNA uptake or electroporation(Shimamoto, et al, 1989, Nature 338, 274-276). Transgenic maize plantshave been obtained by introducing the Streptomyces hygroscopicusbar-gene, which encodes phosphinothricin acetyltransferase (an enzymewhich inactivates the herbicide phosphinothricin), into embryogeniccells of a maize suspension culture by microprojectile bombardment(Gordon-Kamm, 1990, Plant Cell, 2, 603-618). The introduction of geneticmaterial into aleurone protoplasts of other monocot crops such as wheatand barley has been reported (Lee, 1989, Plant Mol. Biol. 13, 21-30).Wheat plants have been regenerated from embryogenic suspension cultureby selecting only the aged compact and nodular embryogenic callustissues for the establishment of the embryogenic suspension cultures(Vasil, 1990 Bio/Technol. 8, 429-434). The combination withtransformation systems for these crops enables the application of thepresent invention to monocots.

[0038] Monocotyledonous plants, including commercially important cropssuch as rice, banana and corn are also amenable to DNA transfer byAgrobacterium strains (vide WO 94/0097; EP 0 159 418 B1; Gould J,Michael D, Hasegawa O, Ulian E C, Peterson G, Smith R H, (1991) Plant.Physiol. 95, 426-434).

[0039] Following DNA transfer and regeneration, putatively transformedplants may be evaluated, for instance using Southern analysis, for thepresence of the recombinant DNA according to the invention, copy numberand/or genomic organization. In addition, or alternatively, expressionlevels of the newly introduced DNA may be undertaken, using Northernand/or Western analysis, techniques well known to persons havingordinary skill in the art. After the initial analysis, which isoptional, transformed plants showing the desired copy number andexpression level of the newly introduced recombinant DNA according tothe invention may be tested for their male sterility or restoration tofertility. Alternatively, the selected plants may be subjected toanother round of transformation, for instance to introduce furthergenes, such as the antisense TPS gene, the TPP gene or the suppressorgene.

[0040] To obtain transgenic plants capable of constitutively expressingmore than one chimeric gene, a number of alternatives are availableincluding the following:

[0041] A. The use of DNA, e.g a T-DNA on a binary plasmid, with a numberof modified genes physically coupled to a selectable marker gene. Theadvantage of this method is that the chimeric genes are physicallycoupled and therfore migrate as a single Mendelian locus.

[0042] B. Cross-pollination of transgenic plants each already capable ofexpressing one or more chimeric genes, preferably coupled to aselectable marker gene, with pollen from a transgenic plant whichcontains one or more chimeric genes coupled to another selectablemarker. Afterwards the seed, which is obtained by this crossing, maybeselected on the basis of the presence of the two selectable markers, oron the basis of the presence of the chimeric genes themselves. Theplants obtained from the selected seeds can afterwards be used forfurther crossing. In principle the chimeric genes are not on a singlelocus and the genes may therfore segregate as independent loci.

[0043] C. The use of a number of a plurality chimeric DNA molecules,e.g. plasmids, each having one or more chimeric genes and a selectablemarker. If the frequency of co-transformation is high, then selection onthe basis of only one marker is sufficient. In other cases, theselection on the basis of more than one marker is preferred.

[0044] D. Consecutive transformation of transgenic plants alreadycontaining a first, second, (etc.), chimeric gene with new chimeric DNA,optionally comprising a selectable marker gene. As in method B. thechimeric genes are in principle not on a single locus and the chimericgenes may therefore segregate as independent loci.

[0045] E. Combinations of the above mentioned strategies.

[0046] Plants, in which this invention is particularly useful, areplants which are able to propagate vegetatively and in which sproutingat a certain moment is an undesired property. The most outstandingexamples are potato and onion, but the invention can also be used inflower bulbs, strawberries and banana. Next to the complete inhibitionof sprouting and an inducible restoration mechanism, it is alsoenvisaged that the inhibition can be made inducible. This, for instance,would be useful in strawberry and banana, where sprouting is a desiredproperty for the multiplication of plants, but where sprouting can becompetitive with regard to other processes such as fruit ripening. Ifthe TPS gene is placed under control of an inducible promoter it ispossible to inhibit sprouting at any time during the growing of thecrops, for instance during the period of seed setting or fruit ripening.Preferably such an induction of expression of the TPS gene is performedby a chemical inducible promoter which reacts on the (external)application of a chemical substance. Furthermore, in this embodiment ofthe invention it would be preferable also to make the expression of TPStissue specific for meristematic tissue. Promoters, which are specificfor meristematic tissue are readily available in the art (for instancethe HMG2 promoter from Enjuto et al., Plant Cell 7, 517, 1995 and therice PCNA promoter from Kosugi et al., Plant J. 7, 877, 1995).

[0047] Next to the sprouting the mechanism of inhibition of pre- andpostharvest remobilisation of storage compounds is also of use inchicory to prevent degradation of inulin and in sugarbeet to preventdegradation of sucrose.

[0048] The following examples are further provided for illustrativepurposes only and are in no way intended to limit the scope of thepresent invention.

[0049] Standard methods for the isolation, manipulation andamplification of DNA, as well as suitable vectors for replication ofrecombinant DNA, suitable bacterium strains, selection markers, mediaand the like are described for instance in Sambrook, J., Fritsch, E. P.,and Maniatis, T. (1989) Molecular cloning; a laboratory manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning:Volumes I and II (D. N. Glover ed. 1985); and in: From Genes To Clones(E.-L. Winnacker ed, 1987).

[0050] DNA Manipulations

[0051] All DNA procedures (DNA isolation from E. coli, restriction,ligation, transformation, etc.) are performed according to standardprotocols (Sambrook et al. (1989) Molecular Cloning: a laboratorymanual, 2nd ed. Cold Spring Harbor Laboratory Press, CSH, New York).

[0052] Strains

[0053] In all examples E. coli K-12 strain DH5α is used for cloning. TheAgrobacterium tumefaciens strains used for plant transformationexperiments are EHA 105 and MOG 101 (Hood et al., Trans. Research 2,208-218, 1993)

[0054] Generation of Potato Plants Transgenic for Pat-TPS.

[0055] Construction of pMOG845 harboring the E. coli tps gene undercontrol of the tuber-specific patatin promoter, triparental mating toAgrobacterium and the generation of transgenic potato plants, Solanumtuberosum cv. kardal, are described in WO 97/42326.

[0056] Experimental Part

EXAMPLE 1

[0057] In one part of the experiment, tuber material was produced fromin vitro potato plants transgenic for pMOG845 (patatin-tps). A fieldtrial experiment was set-up using tubers of 9 independent transgeniclines, 3 plots per line, 5 tubers per plot. Tubers were transferred tothe field at the beginning of May and the sprouting process wasmonitored on a regular basis. Results are depicted in table 1. In thesecond part of the experiment pat-TPS plants (var Kardal) derived fromtissue culture plants were grown in the phytochamber under 500 μmolquanta m-2 s-1 (16 h light, 20_C; 8 h dark (15_C) ). Tubers wereharvested after three months and stored in the cold (4_C) for 2 months.Then they were transferred to room temperature (RT) and sprouting wasassessed during a period of four weeks. TABLE 1 Sprouting Plant-lineField Phytochamber Kardal all tubers all tubers 845-17 all tubers*delayed 845-13 all tubers all tubers 845-28 none none 845-4 all tubersall tubers 845-11 none none 845-22 2/15 tubers none 845-2 all tubers*delayed 845-1 all tubers* delayed 845-25 all tubers all tubers

[0058] Tubers revealing the complete absence of sprouting have beenshown to have a high expression level of the transgene. A reduction ofcold-sweetening as described in WO 97/42326 is observed in thenon-sprouting lines and to a lesser extent in the tubers delayed insprouting or normal sprouting tubers.

EXPERIMENT 2

[0059] Gibberellic Acid Reverts Anti-Sprouting Phenotype

[0060] Whole tubers obtained from the plants of Example 1 grown underphytochamber conditions were taken. Approximately 1 week after transferto RT they were incubated for 24 h in a solution containing 0.17% (w/v)gibberellic acid (GA 4 and GA 7; formulation commercially available asBerelex®, Zeneca, Ridderkerk, Netherlands) Control tubers were notincubated. Further storage was done at RT. The induction of sproutingoccurred in GA-treated and non-treated wildtype tubers after 8 days.After 14 days, 95% of the 14 non-treated wildtype tubers sprouted, whilenone of the transgenic lines did (FIG. 1A). In contrast, all tubers (5)from GA-treated wildtype tubers and 80%, 50%, 100% and 17% of theGA-treated transgenic tubers from lines 845-1, −17, −22, −28 formsprouts, respectively, All non-treated transgenic tubers did not sprout.After 25 days it can be seen that lines 845-1 and 845-17 show delayedsprouting in the non-treated tubers (FIG. 1B).

EXPERIMENT 3

[0061] Wounding Reverts Anti-Sprouting Phenotype

[0062] Pat-TPS plants (Var. Kardal) derived from tissue culture plantswere grown in the phytochamber under 500 μmol quanta m-2 s-1 (16 hlight, 20° C.; 8 h dark (15° C.)). Tubers were harvested after threemonths and stored in the cold (4° C.) for 2 months.

[0063] Three days after transfer to room temperature (RT), tuber pieceswere cut with a knife containing at least one active meristem (eye). Cutpieces originating from 3-10 tubers per line were washed for 15 min intap water. Approximately 6-10 pieces were subsequently incubated for 10min on either water or on a 1, 10 or 1000 ppm solution of gibberellicacid (GA3, SIGMA, Zwijndrecht, Netherlands). All pieces from onetreatment were transferred to containers onto wet paper tissue andcovered with a plastic top to prevent drying out. Sprouting of wild-typeand tps tuber pieces occurred within 4 days incubated either on water oron the different gibberellic acid solutions, indicating that woundingper se is sufficient to restore sprouting.

1. A method to inhibit pre- and/or postharvest remobilisation of storagecompounds in plants by transforming a plant or a plant from its parentalline with a recombinant DNA capable of expression of a protein,characterized in that the protein is trehalose phosphate synthase (TPS).2. A method to prevent sprouting of a plant part by transforming theplant or a plant from its parental line with a recombinant DNA capableof expression of a protein, characterized in that the protein istrehalose phosphate synthase (TPS).
 3. A method according to claim 2,characterized in that the recombinant DNA comprising the gene coding forTPS is of bacterial, fungal, animal, plant or human origin, preferablyderived from Escherichia coli.
 4. Method to induce sprouting in a plantwhich is made non-sprouting according to the method of claim 2 or 3characterized in that said plant is provided with recombinant DNA codingfor TPS flanked by target sites of a site-specific recombinase and thatthe recombinant DNA coding for TPS is removed by providing said planteither through tranformation with a gene coding for the correspondingrecombinase or through crossing with a plant harbouring a recombinantDNA capable of expressing said recombinase.
 5. Method to inducesprouting in a plant which is made non-sprouting according to the methodof claim 2 or 3 by transforming it with a recombinant DNA whichcomprises a gene coding for a compound which is capable of neutralisingthe effects of TPS under control of and inducible promoter and forcingexpression of the TPS neutralizing compound by induction of theinducible promoter.
 6. Method according to claim 5, characterized inthat the neutralising compound is trehalose phosphate (TPP).
 7. Methodaccording to claim 5, characterized in that the neutralising compound isantisense trehalose phosphate synthase.
 8. Method according to claim 5,characterized in that the neutralizing compound is trehalose phosphatehydrolase (TreC).
 9. Method according to claim 5 characterized in thatthe neutralising factor is a suppressor which is capable of suppressingexpression of the TPS.
 10. A method to release the inhibition of pre-and/or postharvest remobilisation of storage compounds in plants causedby the expression of trehalose phosphate synthase, by treating thestorage organ of the plant with gibberellic acid.
 11. Method to inducesprouting in a plant which is made non-sprouting according to the methodof claim 2 or 3 by treating the plant with gibberellic acid.
 12. Methodto induce sprouting in a plant which is made non-sprouting according tothe method of claim 2 or 3 by treating wounding the plant.
 13. Methodaccording to claim 1, characterized in that the storage compound isinulin and the plant is chicory.
 14. Method according to claim 10,characterized in that the storage compound is sucrose and the plant ischicory.
 15. Method according to claim 1, characterized in that thestorage compound is sucrose and the plant is sugarbeet.
 16. Methodaccording to claim 10, characterized in that the storage compound issucrose and the plant is sugarbeet.